A high-resolution solid-state 13C-NMR study on crystalline bovine heart cytochrome-c oxidase and lysozyme. Dynamic behavior of protein and detergent in the complex.

We have recorded 100.6-MHz high-resolution solid-state 13C-NMR spectra of crystalline cytochrome-c oxidase from bovine heart muscle and hen egg-white lysozyme, to compare conformation and dynamics of a typical membrane-protein complex with those of lysozyme. The absence of severe interference with the solid-state 13C-NMR spectra, from both the line broadenings from paramagnetic centers and overlapping of intense detergent signals, provided spectral resolution of 13C-NMR feature of cytochrome-c oxidase crystals comparable to that of lysozyme crystal and better than that of dissolved or lyophilized samples. In fact, the observed peak intensities of the polar heads of the detergents BL8SY and Brij 35 were only about 10% and 3% of the anticipated values, respectively. The dynamic behavior of the backbone and side chains of cytochrome-c oxidase was compared with that of lysozyme on the basis of the 13C spin-lattice relaxation times (T1): the backbone of the cytochrome-c oxidase turned out to be more flexible than that of lysozyme. Molecular motions of the detergent molecules attached to the proteins are found to be highly heterogeneous. Detergent molecules undergo rapid tumbling motions in the crystals in about 10 ns as detected by T1. In addition to rapid motions, slow motions were detected by 1H spin-lattice relaxation time in the rotating frame (TH1 rho) and cross-polarization time (TCH), together with data from static spectra, indicating that the aliphatic portion of the detergent interacts more strongly with hydrophobic protein surfaces than do the polar heads.     

Homologous recombination in bovine papillomavirus shuttlevecter; effect of relative orientation of substrate sequences.

Relative orientation of recombination substrates, neo gene, strongly influenced homologous recombination events in a bovine papillomavirus shuttle vector. Between direct repeats, recombination occurred at a high frequency while between inverted repeats, it was rare. Double strand break near the mutation site increased the recombination frequency between inverted repeats but not between direct repeats. Formation of long heteroduplex as a recombination intermediate may explain this apparently paradoxical phenomenon.     

Phospholipid composition of rat megakaryocytes and its rearrangement in platelets

Rat platelets and their megakaryocyte precursors were examined for phospholipid composition. (1) The phospholipid composition of rat megakaryocytes, which were enriched and prepared from bone marrow cells, was almost identical to that of platelets. (2) The subclass composition of choline-containing glycerophospholipids (CGP) of rat megakaryocytes differed significantly from that of platelets: 1-alkenyl-2-acyl glycerophosphocholine (GPC) in megakaryocytes accounted for 29% of the total, whereas that in platelets was only 7%. (3) Rat platelets contained a larger amount of arachidonic acid than megakaryocytes, especially in ethanolamine-containing glycerophospholipids (EGP). (4) [32P]Phosphoric acid was significantly incorporated into megakaryocytes, whereas platelets showed little incorporation. On the other hand, the uptake of [3H]arachidonic acid into platelet phospholipids was about 15-times higher than that observed with megakaryocytes. (5) As reported previously for other blood cells, such as neutrophils and macrophages, the radioactivity of labeled arachidonic acid incorporated into CGP of platelets decreased, whereas that incorporated into EGP increased during a subsequent chase period. Hardly any such change was observed with megakaryocytes. These results suggest that the phospholipid composition of rat platelets is mainly determined at the time of thrombopoiesis, whereas the composition of molecular species is remodeled during circulation after thrombopoiesis.     

Temperature-jump NMR study of protein folding: Ribonuclease A at low pH

Summary The kinetic process of folding of bovine pancreatic ribonuclease A in a²H₂O environment at pH 1.2 was examined by a recently developed temperature-jump NMR method (Akasaka et al., (1990) Rev. Sci. Instrum.61, 66–68). Upon temperature-jump down from 45°C to 29°C, which was attained within 6 s, the proton NMR spectral changes were followed consecutively in time intervals of seconds. There was a rapid spectral change, which was finished within the jump period, followed by a much slower process which lasted for a minute or longer. Rates of the slower process were measured at different positions of the polypeptide chain as intensity changes of individual His and Tyr proton signals of the folded conformer and as intensity changes of aliphatic and His protons of the unfolded conformer. Most of these rates coincided with each other within experimental error with an average value of 2.8×10^–2s^–1. The result gave clear experimental evidence that the slow folding of RNase A at low pH is a cooperative process involving most regions of the molecule, not only thermodynamically, but kinetically as well.     

Birth of normal calves resulting from bovine oocytes matured, fertilized, and cultured with cumulus cells in vitro up to the blastocyst stage

Bovine oocytes matured in vitro were fertilized in high proportions (92% of matured oocytes) by sperm capacitated with Ca ionophore A23187. Eight percent of inseminated oocytes that were denuded 96 h after insemination developed to the morula stage when cultured for 6-120 h after insemination with cumulus cells from the original oocytes. Inseminated oocytes denuded 96 h after insemination developed to the blastocyst stage when cultured with or without cumulus cells or in the conditioned medium from 96 h to 168-216 h after insemination (9.0%, 8.1%, and 6.8% of inseminated oocytes respectively). Six frozen-thawed blastocysts were transferred nonsurgically to 3 recipients (2 embryos/recipient). Two of the 3 recipients became pregnant, with one delivering live twins at term. Seven fresh blastocysts were transferred nonsurgically to 6 recipients (1-2 embryos/recipient). Three of the 6 recipients became pregnant, with 2 delivering live calves.     

Experimental stomach-ulcer on pika

Experimental gastric ulcer formation was performed in the pika and compared with that in the rat. Gastric ulcers were formed in pika that were subjected to water restraint for 4-5 days for 2 hours each day. Gastric ulcers were also formed under conditions of 1-4 days for 3 hours each day and 1-2 days for 5 hours each day. The severest (widest) ulcers were obtained under the condition of 5 hours' water restraint. Histopathologically, the ulcers were mostly erosions, but those formed under 5 hours' restraint reached the tunica muscularis mucosae. In addition, inflammatory changes were recognized. In contrast, while gastric ulcers in the rat formed within a short time, they were histopathologically less severe than those in the pika. Therefore, water restraint for 4 hours performed 4-5 times is suitable to obtain gastric ulcer formation in the pika and may result in more severe gastric ulcers than in the rat. Compared with the rat, the pika showed differences in the appearance and degree of gastric ulcers formed by the injection of serotonin and reserpine.     

Unfolding and refolding of the constant fragment of the immunoglobulin light chain

The kinetics of reversible unfolding and refolding by guanidine hydrochloride of the constant fragment of the immunoglobulin light chain are described. The kinetic measurements were made at pH 7.5 and 25 °C using tryptophyl fluorescence and farultraviolet circular dichroism.The kinetics of unfolding of the constant fragment showed two phases in the conformational transition zone and a single phase above the transition zone. A double-jump experiment confirmed the presence of two forms of the unfolded molecule. These results were thoroughly explained on the basis of the three-species mechanism, U₁

U₂

N, where U₁ and U₂ are the slow-folding and fast-folding species, respectively, of unfolded protein and N is native protein. The equilibrium constant for the process of U₂ to U₁ was estimated to be about 10 and was independent of the conditions of denaturation. These findings were consistent with the view that the U₁

U₂ reaction is proline isomerization. The rates of interconversion between N and U₂ changed greatly with the concentration of guanidine hydrochloride. On the other hand, the refolding kinetics below the transition zone showed behavior unexpected from the three-species mechanism. Whereas the apparent rate constant of the slow phase of refolding was independent of the refolding conditions, its amplitude decreased markedly with the decrease in the final concentration of guanidine hydrochloride. On the basis of this and other results, formation of an intermediate during refolding was ascertained and the refolding kinetics were consistently explained in terms of a more general mechanism involving a kinetic intermediate probably containing non-native proline isomers. The intermediate seemed to have a folded conformation similar to native protein. Comparison of the refolding kinetics of the constant fragment with those of other domains of the immunoglobulin molecule suggested that Pro143 is responsible for the appearance of the slow phase.     

The binding of ATP to the catalytic and the regulatory site of Ca2+,Mg2+-dependent ATPase of the sarcoplasmic reticulum

Two kinds of ATP binding sites were found on the ATPase molecule in deoxycholic acid-treated sarcoplasmic reticulum. One was the catalytic site (1 mol/mol active site) and its affinity was high. Upon addition of Ca²⁺, all the ATP bound to the catalytic site disappeared at 75 mM KCl, while a significant amount of ATP remained bound to the site at 0–2 mM KCl. The latter binding was found to be due to the formation of a slowly exchanging enzyme-ATP complex, which is in equilibrium with phosphoenzyme + ADP. The other binding site was the regulatory one (1 mol/mol active site) and its affinity was low, changing only insignificantly upon addition of Ca²⁺. The ATP binding to the regulatory site shifted the equilibrium between the slowly exchanging complex and EP toward EP.     

Application of the two-dimensional nmr techniques on a des(Ala,Gly)-somatostatin analog

Two-dimensional nmr techniques have been carried out for the peak assignment of the spectrum of a somatostatin analog. Two-dimensional J-resolved spectroscopy simplified the rather broad and complicated spectrum to show the center of chemical shifts of each resonance and gave information on the coupling profiles. Another technique, two-dimensional spin-echo correlated spectroscopy, revealed the connectivities between protons which are correlated by weak spin–spin couplings. The combination of the results of these two complementary techniques made it possible for us to assign almost all peaks of the spectrum of the 11-residue somatostatin analog.